Methods for ex vivo expansion of natural killer cells and use thereof

Abstract

Provided herein are ex vivo methods for the expansion of cord blood-derived natural killer cells and methods of their use. Examples of embodiments include stimulating mononuclear cells from cord blood in the presence of antigen presenting cells (APCs) and IL-2 and re-stimulating the cells with APCs to produce expanded NK cells. In specific embodiments, the method does not utilize human leukocyte antigen (HLA) matching.

What is claimed is: 
     
         1 . An ex vivo method for the expansion of natural killer (NK) cells comprising:
 (a) obtaining a starting population of mononuclear cells (MNCs) from cord blood;   (b) stimulating the MNCs in the presence of antigen presenting cells (APCs) and IL-2; and   (c) re-stimulating the cells with APCs to produce expanded NK cells, wherein the method is performed in a bioreactor and is good manufacturing practice (GMP) compliant.   
     
     
         2 . The method of  claim 1 , wherein the method further comprises depleting cells positive for CD3. 
     
     
         3 . The method of  claim 2 , wherein the depleting is performed between steps (b) and (c). 
     
     
         4 . The method of  claim 3 , wherein the cells are removed from the bioreactor. 
     
     
         5 . The method of  claim 1 , wherein obtain the starting population of MNCs from cord blood comprises thawing cord blood in the presence of dextran, human serum albumin (HSA), DNAse, and/or magnesium chloride. 
     
     
         6 . The method of  claim 1 , wherein obtain the starting population of MNCs from cord blood comprises thawing cord blood in the presence of dextran and DNase. 
     
     
         7 . The method of  claim 5 , wherein the cord blood is washed in the presence of 10% dextran. 
     
     
         8 . The method of  claim 5  or  7 , wherein the cord blood is suspended in the presence of magnesium chloride. 
     
     
         9 . The method of  claim 8 , wherein the magnesium chloride is at a concentration of 200 mM. 
     
     
         10 . The method of any of  claims 1 - 9 , wherein obtaining comprises performing ficoll density gradient centrifugation to obtain mononuclear cells (MNCs). 
     
     
         11 . The method of any of  claims 1 - 10 , wherein the method does not comprise removal or addition of any media components during step (b). 
     
     
         12 . The method of any of  claims 1 - 11 , wherein the bioreactor is a gas permeable bioreactor. 
     
     
         13 . The method of  claim 12 , wherein the gas permeable bioreactor is G-Rex100M. 
     
     
         14 . The method of  claim 13 , wherein the stimulating of step (b) is performed in 3-5 L of media. 
     
     
         15 . The method of any of  claims 1 - 13 , wherein the APCs are gamma-irradiated. 
     
     
         16 . The method of  claim 15 , wherein the APCs are engineered to express membrane-bound IL-21 (mbIL-21). 
     
     
         17 . The method of any of  claims 1 - 16 , wherein the APCs are engineered to express IL-21, IL-15, IL-7, IL-18, and/or IL-2. 
     
     
         18 . The method of any of  claims 1 - 16 , wherein the MNCs and APCs are cultured at a ratio of 1:2. 
     
     
         19 . The method of any of  claims 1 - 18 , wherein the IL-2 is at a concentration of 50-200 IU/mL. 
     
     
         20 . The method of  claim 19 , wherein the IL-2 is at a concentration of 100 IU/mL. 
     
     
         21 . The method of any of  claims 1 - 20 , wherein the IL-2 is replenished every 2-3 days. 
     
     
         22 . The method of any of  claims 1 - 20 , wherein step (b) is performed for 6-8 days. 
     
     
         23 . The method of  claim 22 , wherein step (b) is performed for 7 days. 
     
     
         24 . The method of any one of  claims 1 - 23 , wherein step (b) does not comprise splitting of the cells. 
     
     
         25 . The method of  claim 24 , wherein the cells are fed twice with IL-2. 
     
     
         26 . The method of any of  claims 1 - 25 , wherein the method comprises the use of 3, 4, 5, or 6 bioreactors. 
     
     
         27 . The method of  claim 26 , wherein the method comprises the use of less than 10 bioreactors. 
     
     
         28 . The method of any of  claims 1 - 27 , wherein the NK cells are expanded at least 500-fold, 800-fold, 1000-fold, 3000-fold, or 5000-fold. 
     
     
         29 . The method of any of  claims 1 - 28 , wherein culturing the NK cells in the bioreactor produces more than 1000-fold NK cells as compared to static liquid culture. 
     
     
         30 . The method of any of  claims 1 - 29 , wherein the method does not comprise human leukocyte antigen (HLA) matching. 
     
     
         31 . The method of  claim 30 , wherein the starting population of NK cells are not obtained from a haploidentical donor. 
     
     
         32 . The method of any of  claims 1 - 31 , wherein the method is performed in less than 15 days. 
     
     
         33 . The method of  claim 32 , wherein the method is performed in 14 days. 
     
     
         34 . The method of any of  claims 1 - 33  wherein the expanded NK cells have enhanced anti-tumor activity as comprises to NK cells expanded from peripheral blood. 
     
     
         35 . The method of  claim 34 , wherein the expanded NK cells have higher expression of cell cycle, cell division, and/or DNA replication genes as compared to NK cells expanded from peripheral blood. 
     
     
         36 . The method of any of  claims 1 - 34 , wherein the expanded NK cells have higher proliferative capacity as compared to NK cells expanded from peripheral blood. 
     
     
         37 . The method of any of  claim 1 - 36 , wherein the expanded NK cells do not exhibit exhaustion. 
     
     
         38 . The method of  claim 37 , wherein exhaustion is detected by measuring expression of perforin, granzyme, CD57, KLRG1, and PD1. 
     
     
         39 . The method of  claim 38 , wherein the expanded NK cells have high expression of perforin and granzyme. 
     
     
         40 . The method of  claim 38 , wherein the expanded NK cells have low or no expression of CD57, KLRG1, and PD1. 
     
     
         41 . The method of any of  claims 1 - 40 , wherein the expanded NK cells comprise a clinically relevant dose. 
     
     
         42 . The method of any of  claims 1 - 41 , wherein the cord blood is frozen cord blood. 
     
     
         43 . The method of  claim 42 , wherein the frozen cord blood has been tested for infectious disease. 
     
     
         44 . The method of any of  claims 1 - 43 , wherein the cord blood is pooled cord blood. 
     
     
         45 . The method of  claim 44 , wherein the cord blood is pooled from 3, 4, 5, 6, 7, or 8 individual cord blood units. 
     
     
         46 . The method of any of  claims 1 - 45 , wherein the NK cells are not autologous. 
     
     
         47 . The method of any of  claims 1 - 46 , wherein the NK cells are not allogeneic. 
     
     
         48 . The method of any of  claims 1 - 47 , wherein the APCs are universal antigen presenting cells (uAPCs). 
     
     
         49 . The method of  claim 48 , wherein the uAPCs are engineered to express (1) CD48 and/or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41BB ligand (41BBL). 
     
     
         50 . The method of  claim 48 , wherein the uAPCs express CD48. 
     
     
         51 . The method of  claim 48 , wherein the uAPCs express CS1. 
     
     
         52 . The method of  claim 48 , wherein the uAPCs express CD48 and CS1. 
     
     
         53 . The method of  claim 48 , wherein the uAPCs have essentially no expression of endogenous HLA class I, II, or CD1d molecules. 
     
     
         54 . The method of  claim 48 , wherein the uAPCs express ICAM-1 (CD54) and LFA-3 (CD58). 
     
     
         55 . The method of any of  claims 1 - 54 , wherein the uAPCs are further defined as leukemia cell-derived aAPCs. 
     
     
         56 . The method of  claim 55 , wherein the leukemia-cell derived aAPCs are further defined as K562 cells. 
     
     
         57 . The method of any of  claims 1 - 56 , further comprising cryopreserving the expanded NK cells. 
     
     
         58 . A pharmaceutical composition comprising a population of NK cells produced by any one of  claims 1 - 57  and a pharmaceutically acceptable carrier. 
     
     
         59 . A composition comprising an effective amount of NK cells produced by any one of  claims 1 - 57  for use in the treatment of a disease or disorder in a subject. 
     
     
         60 . The use of a composition comprising an effective amount of NK cells produced by any one of  claims 1 - 57  for the treatment of an immune-related disorder in a subject. 
     
     
         61 . A method for treating a disease or disorder comprising administering an effective amount of expanded NK cells according to any of  claims 1 - 57  to the subject. 
     
     
         62 . The method of  claim 61 , further comprising administering chemotherapy. 
     
     
         63 . The method of  claim 62 , wherein the chemotherapy is administered prior to the expanded NK cells. 
     
     
         64 . The method of  claim 62  or  63 , wherein the chemotherapy is myeloablative. 
     
     
         65 . The method of  claim 62  or  63 , wherein the chemotherapy is non-myeloablative. 
     
     
         66 . The method of  claim 62  or  63 , wherein the chemotherapy is lymphodepleting chemotherapy. 
     
     
         67 . The method of  claim 66 , wherein the lymphodepleting chemotherapy is fludarabine-based lymphodepleting chemotherapy. 
     
     
         68 . The method of any of  claims 62 - 67 , wherein the chemotherapy is lenalidomide. 
     
     
         69 . The method of any of  claims 62 - 68 , wherein the method does not comprise performing HLA matching. 
     
     
         70 . The method of any of  claims 62 - 69 , wherein the disease or disorder is an immune-related disorder. 
     
     
         71 . The method of  claim 70 , wherein the immune-related disorder is an autoimmune disorder, graft versus host disease, allograft rejection, or inflammatory condition. 
     
     
         72 . The method of any of  claims 61 - 69 , wherein the disease or disorder is cancer. 
     
     
         73 . The method of  claim 72 , wherein the cancer is multiple myeloma. 
     
     
         74 . The method of any of  claims 61 - 73 , wherein the subject does not develop graft versus host disease or other toxicity. 
     
     
         75 . The method of any of  claims 61 - 74 , further comprising administering an effective amount of at least a second therapeutic agent. 
     
     
         75 . The method of  claim 75 , wherein the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiotherapy, or biotherapy. 
     
     
         77 . The method of  claim 75 , wherein the NK cells and/or the at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. 
     
     
         78 . The method of any one of  claims 75 - 77 , wherein the second therapeutic agent comprises elotuzamab.